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Glycogen synthase (GS) activity is reduced in skeletal muscle of type 2 diabetes, despite normal protein expression, consistent with altered GS regulation. Glycogen synthase kinase-3 (GSK-3) is involved in regulation (phosphorylation and deactivation) of GS. To access the potential role of GSK-3 in insulin resistance and reduced GS activity in type 2 diabetes, the expression and activity of GSK-3 were studied in biopsies of vastus lateralis from type 2 and nondiabetic subjects before and after 3-h hyperinsulinemic (300 mU x m(-2) x min(-1))-euglycemic clamps. The specific activity of GSK-3alpha did not differ between nondiabetic and diabetic muscle and was decreased similarly after 3-h insulin infusion. However, protein levels of both alpha and beta isoforms of GSK-3 were elevated (approximately 30%) in diabetic muscle compared with lean (P < 0.01) and weight-matched obese nondiabetic subjects (P < 0.05) and were unchanged by insulin infusion. Thus, both basal and insulin-stimulated total GSK-3 activities were elevated by approximately twofold in diabetic muscle. GSK-3 expression was related to in vivo insulin action, as GSK-3 protein was negatively correlated with maximal insulin-stimulated glucose disposal rates. In summary, GSK-3 protein levels and total activities are 1) elevated in type 2 diabetic muscle independent of obesity and 2) inversely correlated with both GS activity and maximally insulin-stimulated glucose disposal. We conclude that increased GSK-3 expression in diabetic muscle may contribute to the impaired GS activity and skeletal muscle insulin resistance present in type 2 diabetes.

Human skeletal muscle cultures (HSMCs) from type II diabetic subjects were used to determine whether metabolic abnormalities such as hyperglycemia or hyperinsulinemia contribute to the defective muscle glycogen synthase (GS) activity present in this disorder. Following approximately 6 weeks of growth, diabetic cultures were fused for 4 days in normal, hyperglycemia, or hyperinsulinemia medium. Fusion of diabetic HSMCs in hyperglycemia medium (20 mmol/l vs. 5.5 mmol/l) had no effect on GS fractional velocity (FV) or mRNA levels, but impaired acute insulin-stimulation of glycogen synthesis and GS activity at 0.1 mmol/l glucose-6-phosphate, and reduced GS protein content by approximately 15% (P < 0.05). Fusion of diabetic muscle cultures in hyperinsulinemia medium (30 micromol/l vs. 22 pmol/l) improved basal GS activity, increasing the reduced GS FV by approximately 50% (P < 0.05), and decreasing the elevated Km(0.1) (half-maximal substrate concentration) by approximately 47% (P < 0.05). Hyperinsulinemia also significantly increased (P < 0.05) the reduced GS mRNA and protein levels of diabetic muscle to levels similar to that in nondiabetic subjects. In contrast to the improvements in the basal state, hyperinsulinemia completely abolished acute insulin responsiveness of GS activity and glycogen synthesis in muscle of type II diabetic subjects. The combination of hyperinsulinemia and hyperglycemia produced effects on both basal and insulin-responsive GS FV and mRNA similar to hyperinsulinemia alone, but hyperinsulinemia prevented hyperglycemia's effect of lowering GS protein and glycogen synthesis. We concluded that, in diabetic muscle, hyperinsulinemia may serve to partially compensate for the impaired basal GS activity and for the adverse effects of hyperglycemia on GS protein content, activity, and glycogen formation by both pre- and posttranslational mechanisms. Despite these beneficial effects, hyperinsulinemia also induces severe impairment of insulin-stimulated GS activity and glycogen formation, which may contribute to acquired muscle insulin resistance of type II diabetes.

The effects of insulin to stimulate metabolic and mitogenic responses were examined in Rat 1 fibroblast cells that overexpressed either normal (HIRc) or kinase-deficient human insulin receptors. When studied at the optimal growth stage for each cell line, insulin-stimulated responses measured in cells containing kinase-defective receptors with a Lys1018-Ala1018 substitution in the ATP-binding site of the kinase domain (A/K1018). Maximal insulin responsiveness for all these effects, measured as fold-increase over basal, was comparable in parental and HIRc cells (1.8- to 2.4-fold increases). Relative insulin responsiveness for all effects was greatest in A/K 1018 cells. One clone (AK-I) expressing a similar number of kinase-inactive receptors as in the HIRc cells displayed maximal responsiveness of 3.6- to 5.5-fold increases. A second A/K cell line containing 1/10 the number of kinase-inactive receptors displayed responsiveness intermediate between AK-I and parental or HIRc cells (1.5- to 4.8-fold increases). Both clones of kinase-deficient A/K1018 cells displayed impaired insulin sensitivity compared with HIRc cells. These findings suggest that expression of insulin receptor kinase activity is a determinant of insulin sensitivity but not necessarily of the final biological responsiveness of cells to insulin.

We have characterized the insulin-dependent increase in glucose transport in human adipocytes using subcellular fractionation and antibodies specific for the two isoforms of the glucose transporter that are expressed in these cells. Plasma membranes isolated from untreated human fat cells contain the erythroid/GLUT1 isoform of the glucose transporter almost exclusively whereas the muscle-fat/GLUT4 transporter isoform is most abundant in intracellular microsomal membranes in resting cells. After exposure of adipocytes to insulin, the muscle-fat isoform is dramatically increased in the plasma membrane whereas the erythroid isoform barely changes in response to insulin. Thus, the total insulin-mediated increase in plasma membrane glucose transporters, confirmed by affinity labeling of both transporter isoforms, must be due to the increase in the muscle-fat/GLUT4 transporter. The two isoforms exist in different vesicle populations as shown by immunoadsorption of the muscle fat isoform-containing vesicles which are essentially devoid of the erythroid transporter. These data indicate that the insulin-mediated increases in glucose transport in human fat cells is a result of the translocation of vesicles uniquely containing the muscle-fat glucose transporter isoform.

Insulin resistance is a predominant feature in women with polycystic ovarian syndrome (PCO). The cellular mechanisms for this insulin resistance have not been defined. In this study, major steps in the insulin action cascade, receptor binding, kinase activity, and glucose transport activity were evaluated in isolated adipocytes prepared from PCO subjects (n = 8) without acanthosis nigricans and in a group of age and weight-matched controls [normal cycling (NC) n = 8]. The PCO group was hyperinsulinemic and displayed elevated insulin responses to an iv glucose load. The binding of 125I-insulin to adipocytes was similar in cells from PCO and NC subjects. In PCO, autophosphorylation of the insulin receptor-subunit in the absence of insulin was normal but a significant decrease (30% below control) in maximal insulin stimulated autophosphorylation was observed. However, receptor kinase activity measured against the exogenous substrate poly glu:tyr (4:1) was normal. Cells from PCO subjects transported glucose at the same rate, in both the absence and presence of a maximal insulin concentration, as those from NC subjects. Strikingly, there was a large rightward shift in the insulin dose-response curve for transport stimulation in PCO cells (EC50 = 87 +/- 14 pmol in NC vs. 757 +/- 138 in PCO, P less than 0.0005); 8-fold greater insulin concentrations were required to attain comparable glucose transport rates in cells from PCO against NC. In conclusion, our results suggest that insulin resistance in PCO, as assessed in the adipocyte, is accompanied by normal function of insulin receptors, but involves a novel postreceptor defect in the insulin signal transduction chain between the receptor kinase and glucose transport.

Recent results from in vivo studies have shown that the kinetics of insulin action are impaired in lean and obese noninsulin-dependent diabetes mellitus (NIDDM) subjects as well as in obese nondiabetic subjects. We have measured the onset and loss of insulin action on glucose transport in adipocytes obtained from obese nondiabetic and obese NIDDM subjects to determine the contributions of obesity and diabetes to these cellular defects in insulin action. Basal and maximally insulin-stimulated rates of 3-O-methylglucose transport in adipocytes from obese and obese NIDDM subjects were reduced to 50% of the values in cells from normal subjects (P less than 0.05). The activation of glucose transport by insulin (4.3 nmol/L) was slower in cells from obese NIDDM patients. Half of the maximal insulin effect (A50) was reached by 23.0 +/- 5.0 min compared to 9.4 +/- 1.1 min in normal cells (P less than 0.05). Conversely, the deactivation of insulin-stimulated glucose transport upon removal of insulin was more rapid in adipocytes from the obese and obese NIDDM subjects. Half of the maximal insulin effect (D50) was lost by 12.4 +/- 1.7 min in obese NIDDM cells and by 8.9 +/- 1.9 min in obese subjects compared to 25.3 +/- 1.9 min in adipocytes from normal subjects (P less than 0.01). In conclusion, 1) basal and insulin-stimulated rates of glucose transport are similarly reduced in adipocytes from obese and obese NIDDM subjects; and 2) adipocytes from obese and obese NIDDM subjects display defects in the kinetics of insulin action, slower activation and accelerated deactivation, that mirror the defects measured in vivo. Both impairments in the kinetics of insulin action may contribute to the insulin resistance in these subject groups.

We identified a possible endogenous substrate (pp185) of the insulin-receptor kinase in human adipocytes by treating intact cells with insulin and immunoblotting the cellular extracts with polyclonal antiphosphotyrosine antibody. This 185,000-Mr protein was phosphorylated on tyrosine residues in response to insulin in both rat and human adipocytes. The time course of pp185 phosphorylation at 37°C was rapid and corresponded closely to insulin-receptor autophosphorylation but preceded insulin-stimulated glucose transport. Unlike many growth factor receptors, including the insulin receptor, pp185 was not adsorbed to wheat-germ agglutinin. We found that pp185 phosphorylation occurred at 12°C and that the phosphoprotein was associated with both cytoplasmic and membrane fractions at this temperature. Furthermore, pp185 phosphorylation was induced to the same extent as insulin by vanadate and hydrogen peroxide, compounds previously shown to mimic the biologic effects of insulin. In addition, dose-response analysis of insulin-stimulated glucose transport, receptor autophosphorylation, and pp185 phosphorylation resulted in ED50 values of 0.3, 12, and 12 ng/ml, respectively. These results demonstrate the magnitude of “spare” autophosphorylation and pp185 phosphorylation with respect to glucose transport stimulation in human adipocytes. To determine whether the insulin resistance characteristic of non-insulin-dependent diabetes mellitus (NIDDM) and obesity is associated with a defect in receptor autophosphorylation and/or endogenous substrate phosphorylation, we estimated the extent of β-subunit and pp185 phosphorylation in adipocytes from NIDDM, obese, and healthy subjects. Although the efficiency of coupling between receptor activation and pp185 phosphorylation was normal in obesity and NIDDM, the capacity for insulin-receptor autophosphorylation was ∼50% lower in NIDDM subjects compared with nondiabetic obese or lean subjects. Thus, the absolute level of pp185 phosphorylation in response to insulin stimulation would be lower in adipocytes from NIDDM subjects. We conclude that pp185 is directly phosphorylated by the insulin-receptor kinase in vivo and may serve as a mediating step to the biologic effects of insulin in human adipocytes. Furthermore, the extent of pp185 phosphorylation is compromised in adipocytes from NIDDM subjects and may contribute xto the insulin resistance associated with this disorder.

The abilities of insulin and the insulin mimickers spermine and H2O2 to stimulate 3-O-methyl glucose transport in isolated rat ft cells were stuided in an attempt to determine possible common mechanisms of action. All three agents caused a seven- to 12-fold stimulation of initial rates of glucose transport with insulin being the most effective agent. Insulin and spermine displayed similar time courses for the onset of their stimulation of transport; an initial lag before any effect was seen and then a gradual rise until the full effect was reached. The time course of H2O2 activation of glucose transport was different since stimulation was seen at the earliest time point tested and then gradually rose to the maximal effect. Trypsinization of cells removed insulin receptors and rendered the cells insensitive to insulin but not to spermine or H2O2. However, trypsinization did alter the time course of H2O2 action, causing an initial lag phase to appear and a general slowing of the activation kinetics. Pretreatment of cells with 2,4-dinitrophenol to lower ATP levels prevented the stimulatory effects of insulin and the mimickers. All three of the agents revealed a similar temperature dependency for stimulated glucose transport, resulting in linear Arrhenius plots with activation energies of 10.2–11.4 kcal/mole. These results show that (1) H2O2 does not act directly on the glucose transport system of rat adipocytes and (2) insulin and H2O most likely act through a common energy-dependent biochemical pathway to stimulate glucose transport, but H2O2 enters the stimulus-response sequence distal to the initial steps in insulin action.

The ability of temperature to influence insulin binding, hormone stimulation of glucose transport, and the coupling of these two events was studied in isolated rat adipocytes. Insulin binding was reduced as the temperature rose from 16°C to 37°C. Both total and cell surface binding varied in an inverse linear manner with temperature, and Scatchard analysis suggested that this was caused by an altered affinity with no change in receptor number. The insulin responsiveness of 3-O-methyl-glucose transport increased with temperature from a four-fold stimulation at 16°C to 12-fold at 37°C. Insulin sensitivity was also higher at elevated temperatures with the EC50 at 37°C (0.3 ng/mL) one-third that at 16°C (0.9 ng/mL). The stimulation by a single insulin concentration (0.75 ng/mL) varied linearly with temperature. Since the amount of cell-associated insulin that was internalized (non-acid extractable) also increased in a linear fashion with temperature, it was possible to compare the amount of insulin binding to the cell surface needed to attain a certain effect. At 37°C half-maximal stimulation was seen when each cell bound 1255 insulin molecules. The same level of effect required 2510 molecules per cell at 24°C and 6024 molecules per cell at 16°C. These results reveal several things about the insulin-response sequence in fat cells: (1) both total cell and cell surface binding vary inversely with temperature; (2) both insulin responsiveness and sensitivity increase with temperature; (3) the opposing behaviors of binding and sensitivity result in an increase in the effect of a set amount of insulin bound (the coupling efficiency); and (4) the coupling process between insulin receptors and glucose transport is regulated by temperature and could be subject to control by other physiologic factors.

The time course of 125I-insulin dissociation from receptors and deactivation of insulin-stimulated glucose transport was measured in rat adipocytes. When cells were incubated with a submaximally stimulating insulin concentration (1 ng/ml), insulin dissociated rapidly at 24 degrees C and 37 degrees C, with a t1/2 of 26 and 14 min, respectively. On the other hand, deactivation of 3-O-methylglucose transport proceeded at a much slower rate. The t1/2 of deactivation was 73 min at 24 degrees C and 43 min at 37 degrees C. Thus, the activated state of the glucose transport system persisted at a time when receptor occupancy had greatly decreased, and hormone dissociation was 3 times faster than deactivation; both processes were equally temperature-dependent. When glucose (1 mM) was omitted from the buffer, deactivation was essentially completely inhibited for at least 2 h despite the fact that the rate of insulin dissociation was unaffected. In conclusion: 1) termination of the insulin signal on glucose transport can be separated from dissociation of insulin receptor complexes; 2) transport deactivation proceeds much more slowly than insulin dissociation and is dependent on some aspect of ongoing cellular metabolism.

We have studied the ability of occupied insulin receptors to activate (or couple to) the glucose transport system in isolated rat adipocytes. Maximal insulin action is seen when only a small proportion (

In an attempt to elucidate the cellular mechanism(s) of the insulin resistance associated with impaired glucose tolerance (IGT) and non-insulin-dependent diabetes (NIDDM), insulin-sensitive glucose transport was studied employing isolated adipocytes obtained from these subjects using the nonmetabolized glucose analogue, 3-O-methyl glucose. In the subjects with IGT, basal and maximal rates of 3-O-methyl glucose uptake were normal while the responses at submaximal insulin concentrations were decreased, i.e., the dose-response curves were shifted rightward, indicative of decreased insulin sensitivity. In contrast, the dose-response curves for insulin-stimulated 3-O-methyl glucose uptake in adipocytes obtained from subjects with NIDDM were right-shifted and, in addition, there was a marked decrease in both the basal and maximally stimulated rates of glucose transport (i.e., decreased insulin responsiveness). Thus, the adipocytes from subjects with IGT show decreased insulin sensitivity consistent with decreased insulin binding, whereas the adipocytes from subjects with NIDDM exhibit both decreased insulin sensitivity and decreased insulin responsiveness consistent with a combined receptor and postreceptor defect in cellular insulin action. In the groups as a whole, the magnitude of the rightward shift in the dose-response curve for insulin-stimulated glucose transport correlated with the reduction in adipocyte insulin binding (r = 0.47, P < 0.02). In these subjects, the level of fasting hyperglycemia was correlated (P < 0.01) to the magnitude of the decrease in maximal glucose transport and a highly significant correlation was found between the maximal insulinstimulated rate of glucose transport and the maximal in vivo rate of insulin-stimulated glucose disposal (r = 0.49, P < 0.01). Therefore, we conclude that the insulin resistance in subjects with IGT is due solely to a decrease in insulin binding, whereas subjects with NIDDM exhibit both decreased insulin binding and decreased maximal rates of insulin-stimulated adipocyte glucose transport due to a postreceptor defect at this site in the insulin action pathway. If adipocytes are reflective of changes in glucose transport in other insulin target tissues, then these findings suggest that the cellular lesion responsible for the postreceptor defect in insulin action, previously demonstrated in vivo in subjects with NIDDM, resides in part at the level of the glucose transport system.

The potential role of guanine nucleotide regulatory proteins (G-proteins) in acute insulin regulation of glucose transport was investigated by using bacterial toxins which are known to modify these proteins. Cholera-toxin treatment of isolated rat adipocytes had no effect on either 2-deoxyglucose transport or insulin binding. Pertussis-toxin treatment resulted in an inhibition of both insulin binding and glucose transport. Insulin binding was decreased in pertussis-toxin-treated cells by up to 40%, owing to a lowering of the affinity of the receptor for hormone, with no change in hormone internalization. The dose-response curve for insulin stimulation of glucose transport was strongly shifted to the right by pertussis-toxin treatment [EC50 (half-maximally effective insulin concn.) = 0.31 +/- 0.04 ng/ml in control cells; 2.29 +/- 1.0 in treated cells), whereas cholera toxin had only a small effect (EC50 = 0.47 +/- 0.02 ng/ml). Correcting for the change in hormone binding, pertussis toxin was found to decrease the coupling efficiency of occupied receptors (50% of maximal insulin effect with 928 molecules bound/cell in control and 3418 in treated cells). Pertussis-toxin inhibition of insulin sensitivity was slow in onset, requiring 2-3 h for completion. Under conditions where pertussis-toxin inhibition of insulin sensitivity was maximal, a 41,000 Da protein similar to the alpha subunit of Gi (the inhibitory G-protein) was found to be fully ribosylated. These results are consistent with the concept that pertussis-toxin-sensitive G-protein(s) can modify the insulin-receptor/glucose-transport coupling system.

The insulin resistance of type II diabetes mellitus is due to both receptor and postreceptor defects of in vivo insulin action, with the postreceptor defect being the predominant abnormality. Diminished glucose transport has been found in adipocytes from patients with type II diabetes, suggesting that decreased cellular glucose transport activity may be responsible in part for the in vivo postreceptor defect observed in these patients. Recent studies have shown that the in vivo postreceptor defect initially present in patients with Type II diabetes is significantly reversed by insulin therapy. For these reasons, we speculated that the defect in adipocyte glucose transport might also be corrected with exogenous insulin therapy. Therefore, we measured adipocyte 3-O-methylglucose transport in cells from five type II diabetic subjects before and after a 2-week period of intensive insulin treatment. Glycemic control was significantly improved by this regimen. The mean (+/- SE) fasting serum glucose level fell from 292 +/- 24 to 135 +/- 29 mg/100 ml (P less than 0.005), and the mean integrated glucose area under a 7-h meal tolerance test curve decreased from 171,212 +/- 20,403 to 72,408 +/- 9,292 mg/min . dl. The mean 3-O-methylglucose transport activity increased after treatment at all insulin concentrations studied, including basal (before, 0.18 +/- 0.05; after, 0.45 +/- 0.09 pmol/2 X 10(5) cells . 10 sec; P less than 0.005) and maximally effective (25 ng/ml) insulin concentrations (before, 0.50 +/- 0.14; after, 1.32 +/- 0.30 pmol/2 X 10(5) cells . 10 sec; P less than 0.025), although the mean maximal glucose transport activity was still 25% decreased compared to normal values, indicating that a residual in vitro postreceptor defect remained. These results corresponded well with the degree of reversal (75%) of the in vivo postreceptor defect, as assessed by the euglycemic glucose clamp technique. These studies demonstrated that the decrease in adipocyte glucose transport activity in type II diabetes is practically reversible by intensive insulin therapy. This closely corresponds to the reversal by insulin therapy of the postreceptor defect expressed in vivo and provides further evidence that a cellular cause of the postreceptor defect in type II diabetes is a decrease in glucose transport system activity in the major insulin target tissues.

We have studied the effects of the duration of exposure of rat adipocytes to insulin on the temporal relationships between insulin dissociation from receptors and deactivation of insulin-stimulated glucose transport. Cells were incubated with [125I]insulin plus unlabeled insulin at a total insulin concentration of either 1 or 10 ng/ml at 37 C. After various times of association, the cells were washed, and the rate of dissociation of bound hormone and subsequent decrease in insulin-stimulated glucose transport activity (deactivation) were measured. One nanogram of insulin/ml is a submaximally effective concentration and stimulates glucose transport to approximately 80% of maximal values. Dissociation of insulin from receptors and deactivation of glucose transport activity were measured after 10, 30, and 60 min of exposure of cells to 1 ng/ml insulin. Dissociation rates were unchanged after the various times of exposure to insulin, whereas prolonging the preincubation time from 10 to 60 min slowed the rate of deactivation 5-fold. Dissociation and deactivation were also measured after 5-, 10-, 30-, and 60-min periods of incubation with a maximally stimulating hormone concentration (10 ng/ml). The dissociation rates were similar after the various periods of association, whereas the deactivation rate was 2-fold slower after 30 and 60 min of preincubation compared to that after 5 min. Thus, there is a time-dependent slowing of the rate of deactivation of insulin-stimulated glucose transport that is not accounted for by differences in the amount of insulin bound or the dissociation rate of insulin from its receptors. Therefore, the length of exposure to insulin can influence the rate of decline in glucose transport activity, and this may be important in determining biological effects of pulsatile insulin secretion vs. prolonged insulin exposure on insulin target tissues. Furthermore, the data suggest that with time, insulin causes an increase in some intracellular factor or other cellular perturbation, the level of which modulates the rate of deactivation of the glucose transport system.